Changes And Mechanism Of Akt1 In Irradiation-induced Autophagy In Breast Cancer Cells

Currently, breast cancer has become the first serious cancer to harm women's health. This study treated breast cancer cell line MCF-7 with different doses of X-ray and research some genes or proteins expressions on breast cancer related in order to provide a theoretical basis for clinical treatment of breast cancer.Radiotherapy is the most important means of breast cancer treatment. Generally, radiotherapy only is considered to induce apoptosis of tumor cells. In recent years, however, it found that there are cells death occurrence e different from apoptosis because of some process of cells death without occurrence of apoptosis.Autophagy is some external stimuli such as under the conditions of hunger, drug or irradiation to degrade long-lived proteins or organelles and self-recovery process through aut-ophagic lysosomal.Autophagic cell death is a second form of programmed cell death but apoptosis. Changes in autophagy is relative to occurrence and development of cancer.PI3KI/Akt pathway is a signaling pathway of growth factor. PI3KI/Akt activated mTOR kinase. Most of tumors constitutively activated with PI3KI/Akt/mTOR pathway. PI3KI/Akt signaling pathway activated to promote cell survival and proliferation, inhibit apoptosis, control cell cycles, involve in tumor angiogenesis and tumors invasion and metastasis.The article had studied on the effects and roles of MAP1LC3B and Aktl genes in human breast cancer cell line MCF-7. To explore the changes and possible mechanism of PI3KI/Akt pathway established two models of Aktl silencing and Aktl over-expression when autophagy occurred in MCF-7 cell line. It provided a theoretical basis that to search for new cancer treatment programs.1 Changes of MAP1LC3B expression in radiation-induced autophagy in MCF-7 cellsThe method of Realtime RT-PCR was used to detect MAP1LC3B mRNA expression. Dose-response of MAP1LC3B mRNA studies had shown that MAP1LC3B increased in 8,16 and 32h dose-effect studies, up to peak at 12 Gy in 8h study(F=13.831, P＜0.05),12Gy in 16h study (F= 10.996, P＜0.01) and 8Gy in 32h study (F= 17.019, P＜0.01), respectively. Time-response studies have shown that MAP1LC3B mRNA increased and reached to peak at 16h in 2Gy dose-effect study (F=16.284, P＜0.01),32h in 8Gy study (F=9.030, P<0.05)and 16h in 12Gy study (F=20.315, P＜0.05), respectively. The results showed that ionizing radiation can induce autophagy in breast cancer cells.2 Changes of Aktl gene expression in radiation-induced autophagy in MCF-7 cellsThe method of Realtime RT-PCR was used to detect Aktl mRNA expression. Dose-response of MCF-7irradiated groups studies had shown that dose-effect study of Aktl mRNA expression changed in a dose-dependent decreasing at the 2-8Gy interval in 4,8,16h study(F=210.684,555.937,63.766,P<0.01), dose-effect of Aktl expression in 32h study was significantly decreased in a dose-dependent changes, there was a significant difference in 4,8,12 Gy(F=45.461 P＜0.05).Respectively, time-response of MCF-7 cells studies had shown that dose-effect study of Aktl mRNA expression had a significant difference decreased to peak valley at the 8h in 4,8,12Gy study (F=73.059,275.927,152.760,P<0.05), overall of Aktl genes changes were lower than control groups after irradiated.3 Changes of Aktl potein expression in radiation-induced autophagy in MCF-7 cellsThe method of Western blot was used to detect Aktl mRNA expression. The proteins of MCF-7 irradiated groups of studies had shown that Aktl proteins expression had a clear downward trend in 2 Gy time-efect study(decreased 70% compared with control), down to minimum at the 24h(decreased 87.1%). Aktl proteins expression decreased significantly at 0.5h in 4Gy time-efect study(deceased 87.7%), down to minimum at the 8h(decreased 94%). And its expression decreased in a dose-dependent changes trend at 8Gy in 0.5-4h time-efect study, down to minmum at the 4h (decreased 67.3%).4 Establishing of Akt1 silencing and over-expression models in MCF-7 cellsAktl siRNA vectors transfected into packaging cell 293T by using Calcium Phosphate Coprecipitation. After that collected virus soups directly infected MCF-7 cells to establish Aktl silencing model.It were selected to form positive clones by puro and detected Aktl gene expression with Western blot method after the total protein extracted. The results showed that expression of Aktl significantly decreased at 60kD in MCF-7 cells and silencing model was successfully constructed.Plers-Aktl vectors transfected into MCF-7 cell by using lipofection.It were selected to form positive clones by G418 and detected Aktl gene expression with Western blot method after the total protein extracted. The results showed that expression of Aktl significantly increased in MCF-7 cells and over-expression model was successfully constructed.5 MAP1LC3B gene expression in radiation-induced Aktl silencing cells Dose-response of Aktl siRNA model irradiated groups of MCF-7 cells studies had shown that MAP1LC3B mRNA expression increased by varying levels in 8,16,32h dose-efect study and up to peak at the 12 Gy (F=18.181,35.057,21.056,P<0.05). Respectively, time-response of MAPLC3B of Aktl siRNA model cells studies had shown that MAP1LC3B mRNA expression increased significantly at the 16,32h study in 2,4,8Gy (F=19.447,28.405, 7.169, p<0.05), and its expression increased in a dose-dependent at the 12Gy in 8-32h time-efect study, up to maximum at the 32h (F=35.580,P<0.05).6 MAP1LC3B gene expression in radiation-induced Aktl over-expression cellsDose-response of MCF-7-Aktl model irradiated groups of MCF-7 cells studies had shown that overall of MAP 1LC3B mRNA expression decreased to minimum levels in 4,8,16,32h dose-efect study at the 2 Gy (F=849.559,62.132,204.602,48.813, P＜0.05). Respectively, time-response of MAP1LC3B of MCF-7-Aktl model cells studies had shown that MAP1LC3B mRNA expression decreased to peak vally at the 4h study in 2,4,8,12Gy (F=367.355, 564.332,701.623,239.059,P<0.01), and overall of MAP1LC3 expression were lower than control groups after irradiated.In this study, Calcium Phosphate Coprecipitation and lipofection methods were used to constructed Aktl silencing and over-expression models in MCF-7 cells. Realtime RT-PCR and Western blot methods were used to detected the expression of Aktl and/or MAP1LC3B genes.It was shown that X-rays radiation could induce autophagy in MCF-7 cells and futher studied the expression of Aktl gene and protein were shown that autophagy increased in Aktl silencing cells after ionizing radiation, but decreased in Aktl over-expression cells. These results suggested that ionizing radiation may promote autophagy in breast cancer cell line MCF-7 through inhibiting PI3KI/Akt pathway. The conclusions provide foundations for further experimental research, the study of radiotherapy combined with genes therapy in breast cancer cells and a theoretical basis of clinical treatment of breast cancer.